Depending on the gas, measurements made after different breath holding times represent different physiological parameters that change as blood circulates through lungs, arteries, tissues and veins. The clinical utility of existing and future gas measurement devices could be greatly extended by configuring firmware and/or software to record, track and calculate the values in gas measurements made after various breath holding times. The present breath holding methods do not require modifying existing devices, however, beyond selecting the shortest available averaging mode, and can by used anyone simply by holding his or her breath as specified and performing the specified measurements and calculations.
Breath holding time zero [BHt=0] always measures the compartment being sampled. If testing breath, BHt=0 represents the gas level in the lung, if testing blood, BHt=0 represents gas level in the arterial or venous blood at the site drawn, and if testing via skin, BHt=0 represents gas level in whatever organ or tissue type the transcutaneous sensor is placed over, such as an earlobe or fingertip. But beyond BHt=0, the meaning of such measurements is not so clear.
The present invention provides a general method for measuring the relative concentrations of gases that diffuse readily across biological membranes in humans such as carbon monoxide (CO), nitric oxide (NO), hydrogen (H2), and hydrogen sulfide (H2S). The method compares relative levels of the gas from the lungs (L), arteries (A), veins (V) and the average of all tissues (T) by having the subject hold his or her breath for a series of specified times while making repeated or continuous measurements from the same site.
For CO, NO, H2, H2S and other light gases produced endogenously throughout the body in lungs, blood vessels, and/or tissues that also may be inhaled from exogenous sources, breath holding allows the different levels of these gases normally present in different parts of the body to equilibrate within about 35-40 seconds as the blood continues circulating. This is defined as the average of all tissues, T. Shorter breathholding times estimate the levels of gases originally in the lungs (L), arteries (A), and veins (V) when breathholding began.
The differences between the L, A, V and T measures indicate whether a subject is net absorbing the gas (as during current or recent exogenous poisoning), net excreting the gas (as occurs during endogenous poisoning and/or after exogenous poisoning), or within the range of healthy human dynamic equilibrium. Measurements made at breath holding times [BHt] of 0, 4-6, and 20-25 seconds may be the same or different from those made at 35-40 seconds, which estimates the average level in all tissues after they have reached equilibrium with the circulating blood, when T=L=A=V.
In one embodiment of the invention measuring CO via breath, the calculated differences L minus T (L-T or ΔLT) and A minus V (A-V or ΔAV) are used to determine whether the gas is being net inhaled or net exhaled. In another embodiment measuring CO via blood, the calculated differences used to determine net inhalation or exhalation are A-T and T-V, but only one is needed so less painful venous blood samples are recommended.
For all gases measured in breath, at BHt=0 the sample exhaled during the first 2 seconds of a sustained exhalation originates from the mouth and trachea. During the last 2 seconds of exhalation the sample originates from the deepest lung, where most alveoli are and where most gases exchange with blood. This is defined as an end-tidal (ET) sample. These principles have been recognized in pulmonary physiology for centuries. Most FDA-approved breath analyzers specify the measurement of ET samples, while a few specify mouth samples (e.g., measuring H25 in the mouth as an index of halitosis).
What is expected to happen to exhaled gas levels at BHt values greater than zero depends on which gas is being measured, how deeply the subject inhaled before starting breath holding, whether this inhalation was via nose, mouth, or both, and whether the exhalation was via the nose or mouth, and whether the subject was supine (which yields highest ET concentrations]) seated or standing (which yields lowest ET concentrations).
In the present method the subject preferably inhales deeply via the nose before starting to hold his or her breath, as opposed to starting to hold the breath after an exhalation (which is both difficult to do and difficult to replicate), and then exhales via the mouth. The present method requires that a subject consistently either lie flat or sit up with their back as straight as possible during gas sampling.
For primarily inhaled gases such as oxygen (O2) that are always net consumed in tissues, the direction of the overall flow is already known (into the body), but there is still a need to estimate the rate of oxygen transfer from blood into tissues. This is accomplished by measuring the oxygen level at any site by any means at two different BHt and subtracting the difference. The present method recommends BHt=20 and 35 for this purpose since earlier times cannot be used for oxygen because the measured level rises briefly for a few seconds as a result of the immediately preceding deep inhalation and may take up to 10 seconds to reach a short-lived maximum before reversing and gradually declining. This anomaly is not seen if breath holding begins after an exhalation.
For primarily exhaled gases such as carbon dioxide (CO2) that are always net produced in tissues, the direction of overall flow is likewise known (out of the body), and there is a similar need to estimate the rate of carbon dioxide transfer from tissues into blood. As with oxygen, this is accomplished by measuring carbon dioxide level at any site by any means at two different BHt and subtracting the difference. The present method recommends BHt=20 and 35 for this purpose. Earlier times cannot be used for carbon dioxide because the measured level falls briefly for a few seconds as a result of the immediately preceding deep inhalation and may take up to 10 seconds to reach a short-lived minimum before reversing and gradually rising. This anomaly is not seen if breath holding begins after an exhalation.
The differences seen in oxygen and carbon dioxide after breath holding of various times are already recognized as important clinical indexes of lung function, oxygen consumption, and oxidative metabolism, but this is not true of other breath gases such as carbon monoxide, hydrogen sulfide and nitric oxide, for which clinical devices specify only one breath holding time prior to exhalation.
For light gases such as CO, NO, H2, and H2S that circulate in blood and are both produced and consumed in various tissues, and which may be net inhaled or net exhaled depending on the already-circulating level versus the inhaled concentrations, it is impossible to predict whether levels measured at longer breath holding times will rise or fall consistently or remain constant, as they do if already in equilibrium.
The invention is further described using the example of CO but the same principles apply to other gases for which suitable measuring devices are available. Such gases include O2, CO2, NO, NO2, SO2, O3, H2, H2S and ethanol.
Many published studies have looked at the effect of different breath holding times (BHt) on measurements of exhaled CO in parts per million (ppm) with the objective of determining the time required to most closely correlate ET breath levels with venous % COHb. The optimum breath holding time was found to be BHt=20-25 seconds after an initial deep inhalation.
Breath holding also can be done after exhalation but this technique is very difficult to do and replicate so it is not recommended. Breath holding may be done:                a) immediately before discrete gas sampling (e.g. before a blood sample is drawn); or        b) during continuous sampling (e.g. while oximeter is clipped to finger).        
The following publications are representative.
“Effects of acute hypoventilation and hyperventilation on exhaled carbon monoxide measurement in healthy volunteers.” Cavaliere F, Volpe C, Gargaruti R, Poscia A, Di Donato M, Grieco G, Moscato U., BMC Pulm Med. 2009 December 23;9:51. doi: 10.1186/1471-2466-9-51.
“Exhaled carbon monoxide is not flow dependent in children with cystic fibrosis and asthma.” Beck-Ripp J, Latzin P, Griese M., Eur J Med Res. 2004 November 29; 9(11):518-22.
“Validation of the Natus CO-Stat End Tidal Breath Analyzer in children and adults.” Vreman H J, Wong R J, Harmatz P, Fanaroff A A, Berman B, Stevenson D K., J Clin Monit Comput. 1999 December; 15(7-8):421-7
“Evaluation of a fully automated end-tidal carbon monoxide instrument for breath analysis.” Vreman H J, Baxter L M, Stone R T, Stevenson D K., Clin Chem. 1996 January; 42(1):50-6.
“Reproducibility of measurements of trace gas concentrations in expired air.” Strocchi A, Ellis C, Levitt M D., Gastroenterology. 1991 July; 101(1):175-9.
“Acute effect of smoking on rebreathing carbon monoxide, breath-hold carbon monoxide and alveolar oxygen.” Kirkham A J, Guyatt A R, Cumming G., Clin Sci (Lond). 1988 October; 75(4):371-3.
“Is alveolar carbon monoxide an unreliable index of carboxyhaemoglobin changes during smoking in man?” Guyatt A R, Kirkham A J, Mariner D C, Cumming G., Clin Sci (Lond). 1988 January; 74(1):29-36.
“Alveolar carbon monoxide: a comparison of methods of measurement and a study of the effect of change in body posture.” Kirkham A J, Guyatt A R, Cumming G., Clin Sci (Lond). 1988 January; 74(1):23-8.
“First versus second portion of expired air and duration of breath holding in the sampling of expired air carbon monoxide.” Biglan A, Magis K, Dirocco A, Silverblatt A., Br J Addict. 1986 April; 81(2):283-6.
“The effect of duration of breath-holding on expired air carbon monoxide concentration in cigarette smokers.” West R J., Addict Behav. 1984; 9(3):307-9.
“The relationship between alveolar and blood carbon monoxide concentrations during breathholding; simple estimation of COHb saturation.” Jones R H, Ellicott M F, Cadigan J B, Gaensler E A., J Lab Clin Med. 1958 April; 51(4):553-64.
“A new method for rapid precise determination of carbon monoxide in blood.” Gaensler E A, Cadigan J B Jr, Ellicott M F, Jones R H, Marks A., J Lab Clin Med. 1957 June; 49(6):945-57.
“The absorption of carbon monoxide by the lungs during breath-holding.” Forster R E, Fowler W S, Bates D V, Van Lingen N B., J Clin Invest. 1954 August; 33(8):1135-45.
From these studies of CO measured in ET samples exhaled via mouth it is known that:                1) samples collected at BHt=0 measure the level of CO in the lung at rest;        2) samples collected at BHt=20-25 seconds correlate most closely with the percent (%) of carboxyhemoglobin (COHb) in venous blood; and        3) in subjects with recent CO exposure whose internal CO levels have not yet returned to equilibrium, shorter and longer breath holding times give lower CO levels than does a BHt=20-25. The authors interpreted these differences in various ways but never as estimates of either the arterial gas level or the average level in all tissues.        
Other studies show that, after hours of continuous exposure to any fixed level of CO, subjects reach dynamic equilibrium as the CO they are inhaling first saturates their lungs, then their arterial blood, then their tissues, and lastly their venous blood up to the same level as in air.
Similarly, in fresh air when the CO level is zero, healthy subjects in equilibrium with this environment will exhale zero CO at all breath holding times from 0 to 40 s. So as BHt extends beyond 25 s the ET CO concentration may stay the same if venous blood and tissues are already in equilibrium. It will fall only if the average level in tissues is now lower than that in venous blood (suggesting a relatively brief period of CO exposure that did not saturate tissues). It will rise only if the level in tissues is still higher due to more CO accumulating in tissues from higher than normal endogenous sources and/or impaired CO metabolism, and/or prior CO poisoning.
Because free CO (that which is unbound to hemoglobin) diffuses readily through capillaries just as free oxygen does, it does not take long for CO to reach this equilibrium point during breath holding. By my own experiments and consistent with results published by others, it takes BHt=35-40 for CO to equilibrate.
Thus I conclude that:                4) samples collected at or after BHt=35-40 s represent the average equilibrium concentration of all tissues; and        5) a BHt that best represents arterial CO levels exists and falls between that used to measure lung (BHt=0) and venous blood (BHt=20-25)        
Conclusion (4) can be checked by taking both arterial and venous COHb samples at or after BHt=35-40 s. These values should match closely if the average CO level in tissues is really now in equilibrium with both venous and arterial blood.
However during constant exposure to any given CO level it usually takes at least several hours for ET samples to reach equilibrium with inhaled levels. The lower the CO exposure concentration, the longer the time to equilibrium when the level of CO in air>lung>arteries >tissue>veins. Only at and beyond equilibrium are these levels all equal and not changed by any breath holding time.
This fact can be seen easily in healthy people who have no excess CO in their bodies and who, when in equilibrium with fresh air (CO=zero), have the same zero level of CO in their exhaled breath at all breath holding times. However, when put in an exposure chamber with 100 ppm CO, as was commonly done in the 1970 s, they eventually reach equilibrium with the environment so that air=lung=arterial=tissue=venous=100 ppm.
Conclusion (5) proposes that a BHt value exists that represents arterial blood CO levels, and that time value falls between lung (BHt=0) and venous blood (BHt=20-25). The basis for this assertion follows.
First, there is no difference in CO at any BHt for people in equilibrium, so we must consider cases not in equilibrium. During exposure to significant exogenous CO, measurements show the lung CO>average tissue and arterial CO>venous CO, which indicates net absorption of CO from the lungs into the body. After significant exogenous CO poisoning ends and until equilibrium with CO-free air is regained, tissue CO>lung and venous CO>arterial CO. This indicates net excretion of CO from the body.
From the direction of blood flow it seems that any BHt correlating with arterial COHb, if it exists, must fall between the BHt associated with the lung fraction and that associated with the venous fraction. This means in the range of 3 to 17 seconds in non-smokers, given that BHt=0 represents lung CO, and BHt of 20-25 represents venous CO.
In preliminary testing, all subjects were in equilibrium with values of BHt=0 or 1; immediately following a brief CO exposure, subjects showed increases in ET CO at all BHt.
BHt=5 s appears to represent arterial CO levels since this is the time at which a small decrease can be seen which reverses by BHt=10 s and which becomes a steep increase by BHt=15 s, eventually peaking in the BHt=20-25 s venous range.
CO concentration measured around 5 seconds after the start of breath holding was:                a) consistently lower than that measured at BHt=0 if the lung started with a higher level of CO than the arterial blood (into which it diffuses); but        b) consistently higher than that measured at BHt=0 if the lung started with a lower CO level than in arterial blood (into which it could not diffuse).        
This is why BHt=5 (+/−1) seconds (about 5 seconds) represents arterial levels better than 10 or 15 seconds.
Values representing lung (L), arteries (A), venous (V) and tissue (T) can be used to calculate the following clinically significant relationships, where T=average equilibrium of CO in all tissues.
ΔAV=net CO in [arteries—veins]
ΔLT=net CO in [lungs—average of all tissues]
ΔAT=net CO in [arteries—average of all tissues]
ΔVT=net CO in [veins—average of all tissues]
All of these gaps may be negative, positive or equal. When all these gaps are positive, the results indicate net CO uptake into tissues; when all negative, net CO excretion from tissues; and when equal, net equilibrium between air, blood and tissues. In the rare cases when some of these gaps are positive and some negative, the subject's CO status is in flux and can only be interpreted on a case-by-case basis after retesting.